Project Summary Open reading frames encoding proteins smaller than 60 amino acids are ubiquitous in bacteria, with sequence analyses suggesting perhaps hundreds expressed per bacterial genome. Functional studies of the encoded small proteins are limited, but available evidence suggests that they play important roles in the modulation of cell functions including cell division, signal transduction, and nutrient transport. One group of small proteins are the so-called toxins of Type I toxin-antitoxin systems (TA-1). Originally identified on bacterial plasmids, they were determined to function as stability determinants programming the cell death of plasmid- free segregants. Sequence analysis later identified thousands of TA-1 systems on bacterial chromosomes where their functions remain mostly unknown. Because of their known plasmid functions and the presence of putative transmembrane domains, it was assumed that the TA-1 ?toxins? killed or inhibited cell growth under conditions of stress by directly compromising the integrity of the cell membrane. This conclusion was supported by the identification of one system, TisB/IstR, in which the ?toxin? depolarizes membranes and puts the cells in stasis resulting in persistence and tolerance to antibiotics. However, recent data suggests that not all TA-1 ?toxins? disrupt membrane integrity or are toxic when over-produced, suggesting that our current view of these systems is over-simplified. We hypothesize instead that many TA-1 ?toxins? perform subtle cellular functions that do not necessarily involve inhibition of cell growth. To test this hypothesis we will study two evolutionarily related TA-1 ?toxins? in the opportunistic pathogen Enterococcus faecalis: FstpAD1, a plasmid- encoded TA-1 toxin playing a classical role in replicon stabilization, and FstEF0409, a chromosomally encoded toxin of unknown function. The gene for FstEF0409 is linked to genes required for growth in mannitol and preliminary evidence indicates that this ?toxin? modulates carbon flux in E. faecalis. In spite of sequence similarities, over-expression of FstpAD1 and FstEF0409 has differential effects on the cellular transcriptome, suggesting they have distinct target interactions. In addition, cells grown in mannitol-containing medium are much more sensitive to expression of both Fst proteins. Experiments are proposed that will 1) take advantage of the differential activities of the two Fst proteins to determine the amino acid signatures responsible and 2) take advantage of the mannitol induced hypersensitivity to identify the target of the Fst proteins. The results of these experiments will lay the groundwork for detailed interaction and functional analyses of Fst activity. Since these proteins are members of a family of TA-1 ?toxins? that are widely distributed among Gram-positive bacteria, it is likely that what is learned will broadly impact our knowledge of TA-1 system function.